The study of gut microbiota (intestinal microbes) in the human body has become a new frontier for medical discovery. Emerging evidence highlights gut microbiota as a metabolically active 'organ' and novel source of risk factors for cardiovascular disease. Recently, using metabolomics approaches, Stanley Hazen and colleagues (collaborators on the proposed study) have identified several gut microbiota-dependent metabolites from dietary components such as choline and red meat that contribute to the pathogenesis of atherosclerosis and are linked to cardiovascular events in humans. This competing renewal grant aims to test new hypotheses on the basis of these highly promising preliminary findings, and to provide novel insights into the role of gut flora metabolites in the development of coronary heart disease (CHD). We propose a cost-efficient investigation in the Nurses' Health Study (NHS) cohort that will use archived fasting blood specimens from participants collected at two time points approximately 10 years apart. In a nested case-control study including 425 incident CHD cases and 425 matched controls, we will examine: 1) whether plasma concentrations of gut microbiota-dependent metabolites (trimethylamine N-oxide [TMAO], choline, and L-carnitine) are associated with subsequent CHD risk; 2) whether 10-year changes in, or cumulative exposure to, these metabolites predict incident CHD; 3) whether gut microbiota metabolites mediate the cardiovascular risks associated with specific dietary factors such as choline and red meat; and 4) whether blood lipids and inflammatory markers mediate the association between gut flora metabolites and CHD risk. Our study has major strengths including a prospective and highly cost-efficient design, long-term follow-up, repeated collection of blood samples, well-documented incident CHD cases, leveraging of a wide array of previously measured cardio-metabolic biomarkers in our plasma samples, and longitudinal information on diet and lifestyle. This competing renewal builds upon our long-standing research in NHS on risk factors for CHD in women (resulting in 286 publications over the past 30 years) and extends our work to the study of novel risk factors derived from gut microbiota. The findings of our proposed project may elucidate new etiologic pathways for atherothrombotic vascular disease, as well as provide novel prevention options and therapeutic strategies for cardiovascular diseases (including diet/lifestyle, probiotic, and targeted pharmacologic approaches). Thus, the proposed study has the potential for major clinical and public health impact.